Accessibility of Four Arginine Residues on the S4 Segment of the Bacillus halodurans Sodium Channel

Accessibility of Four Arginine Residues on the S4 Segment of the Bacillus halodurans Sodium Channel
Blanchet, Jonathan; Chahine, Mohamed
2007-06-14 00:00:00
The voltage-gated Na+ channel of Bacillus halodurans (NaChBac) is composed of six transmembrane segments (S1–S6), with a pore-forming region composed of segments S5 and S6 and a voltage-sensing domain composed of segments S1–S4. The S4 segment forms the core of the voltage sensor. We explored the accessibility of four arginine residues on the S4 segment of NaChBac, which are positioned at every third position from each other. These arginine residues on the S4 segment were replaced with cysteines using site-directed mutagenesis. Na+ currents were recorded using the whole-cell configuration of the patch-clamp technique. We tested the effect of the sulfhydryl reagents applied from inside and outside the cellular space in the open and closed conformations. Structural models of the voltage sensor of NaChBac were constructed based on the recently crystallized KvAP and Kv1.2 K+ channels to visualize arginine residue accessibility. Our results suggest that arginine accessibility did not change significantly between the open and closed conformations, supporting the idea of a small movement of the S4 segment during gating. Molecular modeling of the closed conformation also supported a small movement of S4, which is mainly characterized by a rotation and a tilt along the periphery of the pore. Interestingly, the second arginine residue of the S4 segment (R114) was accessible to sulfhydryl reagents from both sides of the membrane in the closed conformation and, based on our model, seemed to be at the junction of the intracellular and extracellular water crevices.
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Accessibility of Four Arginine Residues on the S4 Segment of the Bacillus halodurans Sodium Channel